The Centers for Disease Control and Prevention (CDC) estimate that, in the United States, there are in excess of 75 million illnesses per year, 300,000 hospitalizations, and 5,000 deaths annually that are directly attributed to food-borne pathogens [1]. E. coli O157:H7, Listeria monocytogenes, Salmonella enterica and Cryptosporidium parvum food and water borne pathogens are accountable for 1 million infections annually [1]. The CDC estimates that infections from these organisms costs the country 1 billion dollars per year and 5 billion dollars total for all food and water related illness. Thus a major challenge to our public health system is the prevention of infections resulting from food- and water-borne pathogens. The goal of this project is to develop a method for the sensitive detection and identification of infectious agents (bacteria, viruses, and protozoan parasites) associated with food and water-borne diseases. In Phase 1, we intend to develop a fluorescent, multiplex, one-tube method based upon coupling SNIPase probes and rolling circle technology to selectively recognize defined target sequences that are present in the genome of specific pathogenic microorganisms of interest. In the first phase of this project we propose to: Combine SNIPase probes to Rolling Circle Amplification technology using target DNA sequences from E. coli O157:H7 as a model. Demonstrate the feasibility of combining rolling circle technology and SNIPase probes in to discriminate between E. coli O157:H7 and other closely related strains. Show the feasibility of detecting pathogen sequences in fluorescent multiplex assay formats. Neither SNIPase probes nor rolling circle amplification technology by themselves have the necessary sensitivity to detect low levels of DNA targets. By coupling the two reactions we anticipate that we can achieve greater than a billion fold amplification in a relatively short period of time. Additionally due to the inherent nature of the technology in that there is not an abundance of primers that have 3'OH ends, therefore we can eliminate low signal to ratios as a result of primer dimer formation as well background associated with the mis-priming and subsequent DNA synthesis associated with other multiplex technologies.